This study uses QbD and Box-Behnken design to validate and optimize RP-HPLC parameters for irinotecan hydrochloride with the goal of achieving short retention time. The method used a C18 column and a mobile phase of acetonitrile and potassium dihydrogen phosphate (pH 3.2). Retention time was 2.16 min, with linearity from 40–120 µg/ml. LOD and LOQ were 0.8 ng/ml and 2.0 ng/ml. The method was validated per ICH guidelines. Stability studies confirmed a 5-day stability period for analyte and standard.